The polymerase chain reaction (PCR) permits rapid amplification of a nucleic acid having an intervening nucleic acid sequence flanked by known sequences. In PCR, the known sequence information is used to design single-stranded primers which will hybridize to the nucleic acid. After annealing, the primers are extended by a polymerase. The extended products are then removed from the nucleic acid so that a new cycle of annealing and extension can be performed. In performing successive cycles of annealing and extension, the nucleic acid is thereby amplified.
PCR also allows easy cloning of any nucleic acid sequence flanked by known sequences by designing primers which contain restriction endonuclease cleavage sequences. After amplification, the double-stranded PCR product can be digested with the corresponding endonucleases to obtain small 5' or 3' overhang sequences characteristic of the endonucleases. The PCR products are ready for cloning into a vector digested with the same endonucleases. However, this method is limited by the often inefficient cleavage of restriction endonuclease cleavage sites near an end of a double-stranded nucleic acid.